Stem cell cultures

ABSTRACT

The present invention relates compounds for stabilizing cells and methods of their use.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/132,379, filed Jun. 2, 2011, which was filed under 35 U.S.C. §371from PCT Application No. PCT/US2009/066554, filed Dec. 3, 2009, whichclaims priority to U.S. Provisional Application No. 61/200,808, filedDec. 3, 2008, each of which is incorporated in its entirety herein forall purposes.

BACKGROUND OF THE INVENTION

Embryonic stem cells (ESCs) are pluripotent cells that have the capacityto self-renew indefinitely and to differentiate into all cell types ofthe body (Thomson, J. A. et al., Science 282 (5391): 1145-1147 (1998);Thomson, J. A. & Odorico, J. S., Trends Biotechnol 18 (2):53-57 (2000)).This ability provides hope that ESCs will one day be used to replacelost and damaged cells, and provide therapies beyond the reach ofconventional drugs. However, to fully realize the clinical potentials ofhESCs, chemically-defined, feeder- and animal product-free, robustculture conditions have to be established. Although severalchemically-defined media have been reported (Yao, S. et al., Proc NatlAcad Sci USA 103 (18):6907-6912 (2006); Lu, J. et al., Proc Natl AcadSci USA 103 (15):5688-5693 (2006); Ludwig, T. E. et al., Nat Biotechnol24 (2):185-187 (2006)), they are still largely unsatisfactory due to thesuboptimal performance of cells in them. Especially under theseconditions, when cells are passaged by trypsin to single cells, theyundergo extensive cell death. A number of signaling pathways thatmediate hESC self-renewal are known, including FGF, TGF-β, Wnt, etc.(James, D. et al., Development 132 (6):1273-1282 (2005); Xu, R. H. etal., Nat Methods 2 (3):185-190 (2005); Beattie, G. M. et al., Stem Cells23 (4):489-495 (2005); Greber, B., Lehrach, H., & Adjaye, J., Stem Cells25 (2):455-464 (2007); Sato, N. et al., Nat Med 10 (1):55-63 (2004)).However, none of them appears to act as a survival factor in thisprocess, the molecular mechanism of which being elusive.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compounds having the formula:

whereinring A is a substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl;ring B is a substituted or unsubstituted heterocycloalkyl, orsubstituted or unsubstituted heteroaryl;L¹ is —C(O)—NR²— or —C(O)—NR²—;L² is a bond, substituted or unsubstituted alkylene or substituted orunsubstituted heteroalkylene; andR¹ and R² are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In some embodiments, ring A is a substituted or unsubstituted aryl.

In some embodiments, ring A is a substituted or unsubstituted phenyl.

In some embodiments, ring B is a substituted or unsubstitutedheterocycloalkyl, or substituted or unsubstituted heteroaryl.

In some embodiments, ring B is a substituted or unsubstitutedheteroaryl.

In some embodiments, ring B is a substituted or unsubstituted pyrazolyl,substituted or unsubstituted furanyl, substituted or unsubstitutedimidazolyl, substituted or unsubstituted isoxazolyl, substituted orunsubstituted oxadiazolyl, substituted or unsubstituted oxazolyl,substituted or unsubstituted pyrrolyl, substituted or unsubstitutedpyridyl, substituted or unsubstituted pyrimidyl, substituted orunsubstituted pyridazinyl, substituted or unsubstituted thiazolyl,substituted or unsubstituted triazolyl, substituted or unsubstitutedthienyl, substituted or unsubstituted dihydrothieno-pyrazolyl,substituted or unsubstituted thianaphthenyl, substituted orunsubstituted carbazolyl, substituted or unsubstituted benzothienyl,substituted or unsubstituted benzofuranyl, substituted or unsubstitutedindolyl, substituted or unsubstituted quinolinyl, substituted orunsubstituted benzotriazolyl, substituted or unsubstitutedbenzothiazolyl, substituted or unsubstituted benzooxazolyl, substitutedor unsubstituted benzimidazolyl, substituted or unsubstitutedisoquinolinyl, substituted or unsubstituted isoindolyl, substituted orunsubstituted acridinyl, substituted or unsubstituted benzoisazolyl, orsubstituted or unsubstituted dimethylhydantoin.

In some embodiments, L² is substituted or unsubstituted C₁-C₁₀ alkyl.

In some embodiments, L² is unsubstituted C₁-C₁₀ alkyl.

In some embodiments, L² is methylene.

In some embodiments, ring A is substituted or unsubstituted aryl; ring Bis substituted or unsubstituted heteroaryl; R¹ is hydrogen; and L² isunsubstituted C₁-C₁₀ alkyl.

In some embodiments, R² is hydrogen.

In some embodiments, R¹ is hydrogen or unsubstituted C₁-C₁₀ alkyl.

In some embodiments, R¹ is hydrogen.

In some embodiments, the compound has the formula:

wherein, y is an integer from 0 to 3; z is an integer from 0 to 5; X is—N═, —CH— or —CR⁵═; R³, R⁴ and R⁵ are independently CN, S(O)nR⁶, NR⁷R⁸,C(O)R⁹, NR¹⁰—C(O)R¹¹, NR¹²—C(O)—OR¹³, —C(O)NR¹⁴R¹⁵, —NR¹⁶S(O)2R¹⁷,—OR¹⁸, —S(O)2NR¹⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, whereinn is an integer from 0 to 2, wherein if z is greater than 1, two R³moieties are optionally joined together to form a substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; and R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷,R¹⁸ and R¹⁹ are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.

In some embodiments, L² is substituted or unsubstituted C₁-C₁₀ alkyl.

In some embodiments, L² is unsubstituted C₁-C₁₀ alkyl.

In some embodiments, L² is methylene.

In some embodiments, X is —N═ or —CH═.

In some embodiments, z is 2 and two R³ moieties at adjacent vertices arejoined together to from a substituted or unsubstituted heterocycloalkyl.

In some embodiments, z is 1.

In some embodiments, y is 0 or 1.

In some embodiments, R³ is —OR¹⁸, and R¹⁸ is hydrogen or unsubstitutedC₁-C₁₀ alkyl.

In some embodiments, L² is methylene; X is —N═ or —CH═; R¹ is hydrogen;and y and z are 0.

In some embodiments, the compound has the formula:

The present invention also provides for compounds having the formula:

wherein

-   -   Ring D is substituted or unsubstituted aryl or substituted or        unsubstituted heteroaryl;    -   L³ is —C(O)NH— or —S(O)₂NH—;    -   R²⁰ is substituted or unsubstituted alkyl, substituted or        unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   R²¹ is —NR²²R²³ or —OR²⁴;    -   R²² and R²³ are independently hydrogen, substituted or        unsubstituted alkyl, substituted or unsubstituted heteroalkyl,        substituted or unsubstituted cycloalkyl, substituted or        unsubstituted heterocycloalkyl, substituted or unsubstituted        aryl, substituted or unsubstituted heteroaryl, or joined        together to form a substituted or unsubstituted heterocycloalkyl        or substituted or unsubstituted heteroaryl;    -   R²⁴ is substituted or unsubstituted alkyl, substituted or        unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, substituted or unsubstituted        heteroaryl, or joined together to form a substituted or        unsubstituted cycloalkyl of substituted or unsubstituted        heterocycloalkyl.

In some embodiments, ring D is substituted or unsubstituted phenyl.

In some embodiments, R²⁰ is substituted or unsubstituted alkyl orsubstituted or unsubstituted cycloalkyl.

In some embodiments, R²⁰ is substituted or unsubstituted C¹-C¹⁰ alkyl orsubstituted or unsubstituted 3 to 7 membered cycloalkyl.

In some embodiments, R²⁰ is substituted or unsubstituted C¹-C⁵ alkyl orsubstituted or unsubstituted 3 to 6 membered cycloalkyl.

In some embodiments, R²⁰ is unsubstituted C¹-C⁵ alkyl or unsubstituted 3to 6 membered cycloalkyl.

In some embodiments, R²² is hydrogen; and R²³ is substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In some embodiments, R²² is hydrogen; and R²³ is substituted orunsubstituted substituted or unsubstituted aryl.

In some embodiments, R²² and R²³ are joined together to from asubstituted or unsubstituted heterocycloalkyl, or substituted orunsubstituted heteroaryl.

In some embodiments, R²² and R²³ are joined together to from asubstituted or unsubstituted pyrrolyl, substituted or unsubstitutedisoindolinyl, substituted or unsubstituted piperidinyl, or substitutedor unsubstituted tetrahydroquinolinyl.

In some embodiments, compound has the formula

wherein, w is an integer from 0 to 1; q is an integer from 0 to 7; R²⁵,R²⁶, R²⁷ and R²⁸ are independently —CN, —NR²⁹R³⁰, —C(O)R³¹,—NR³²—C(O)R³³, —NR³⁴—C(O)—OR³⁵, —C(O)NR³⁶R³⁷, —NR³⁸S(O)₂R³⁹, —OR⁴⁰,—S(O)₂NR⁴¹, —S(O)_(v)R⁴², substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl,wherein v is an integer from 0 to 2; R²⁹, R³⁰, R³¹, R³², R³³, R³⁴, R³⁵,R³⁶, R³⁷, R³⁸, R³⁹, R⁴⁰, R⁴¹, and R⁴² are independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl, wherein R²⁵ and R²⁶, or R²⁶ and R²⁷, may bejoined to form a substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In some embodiments, R²⁸ is —OR⁴⁰, wherein R⁴⁰ is hydrogen orunsubstituted C₁-C₁₀ alkyl.

In some embodiments, R⁴⁰ is hydrogen or unsubstituted C₁ to C₅ alkyl.

In some embodiments, the compound has the formula:

In some embodiments, R²⁰ is unsubstituted C₁ to C₁₀ alkyl.

In some embodiments, R²⁸ is —OR⁴⁰, wherein R⁴⁰ is hydrogen orunsubstituted C₁ to C₁₀ alkyl, or C₁ to C₁₀ alkyl substituted withsubstituted or unsubstituted C₃ to C₆ cycloalkyl.

In some embodiments, q is 1.

In some embodiments, the compound has the formula:

In some embodiments the compounds have the formula:

The present invention also provides methods of stabilizing an isolatedcell in vitro. In some embodiments, the method comprises contacting ananimal cell with a sufficient amount of a compound of formula I or IIIto stabilize the cell.

In some embodiments, the method further comprises changing theconditions or environment of the cell in the presence of the compound,wherein the changing step in the absence of the compound would result ina change in the cell's cellular programming. In some embodiments, thechanging step comprises at least one of thawing the cells anddissociating the cells from other cells.

In some embodiments, the cell is adherent. In some embodiments, the cellis in suspension.

In some embodiments, the method further comprises determining aphenotype of the cell.

In some embodiments, the method comprises isolating the cells from ananimal. In some embodiments, the animal is a human. In some embodiments,the animal is a non-human animal.

In some embodiments, the compound is a compound of formula I. In someembodiments, the compound is a compound of formula III.

The present invention also provides methods of ameliorating a conditionin an animal. In some embodiments, the method comprises administering asufficient amount of a compound of formula I or III to an animal in needthereof to ameliorate the condition.

In some embodiments, the condition is selected from the group consistingof tissue damage, stroke, and cancer. In some embodiments, the tissue isselected from the group consisting of pancreas, liver, intestine, lung,and kidney.

In some embodiments, the condition comprises at least partial rejectionof a transplanted tissue or organ. In some embodiments, thetransplantation comprises transplantation of bone marrow, cord blood,purified hematopoietic stem or progenitor cells, cardiac cells, neuralcells, pancreatic beta cells, or liver cells.

In some embodiments, the compound is a compound of formula I. In someembodiments, the compound is a compound of formula III.

The present invention also provides methods for maintaining cellsurvival. In some embodiments, the method comprises generating isolatedstem cells, progenitor cells, or differentiated cells; and inducingstabilization of E-cadherin in the isolated cells, thereby maintainingcell survival.

In some embodiments, the inducing step comprises contacting the isolatedstem cell with an amount of a compound of formula I sufficient toimprove survival of isolated stem cells by at least 2-fold compared tothe absence of the compound.

In some embodiments, the inducing step comprises culturing the isolatedstem cells on a surface, wherein a molecule comprising an E-Cadherinectodomain is tethered to the surface.

The present invention also provides populations of isolated cellscomprising an amount of a molecule that stabilizes E-cadherin in thecells sufficient to improve survival of isolated cells by at least2-fold compared to the absence of the molecule.

In some embodiments, the molecule comprises a compound of formula I.

In some embodiments, the cells are selected from the group consisting ofstem cells, induced stem cells, pluripotent stem cells, progenitorcells, differentiated cells, beta cells and fibroblasts.

The present invention also provides for populations of isolated cellscomprising an amount of a compound of formula I or III sufficient toimprove survival of isolated cells by at least 2-fold compared to theabsence of the compound.

In some embodiments, the cells are selected from the group consisting ofstem cells, induced stem cells, pluripotent stem cells, progenitorcells, differentiated cells, beta cells and fibroblasts.

The present invention also provides methods for maintaining stem cellsurvival. In some embodiments, the methods comprise generating isolatedcells; and activating protein kinase C (PKC) in the isolated cells,thereby maintaining cell survival.

In some embodiments, the activating step comprises contacting asufficient amount of phorbol 12-myristate 13-acetate (PMA) to theisolated cells to improve survival of the cells compared to the survivalrate in the absence of PMA.

The present invention also provides populations of isolated stem cellscomprising an amount of protein kinase C activator sufficient to improvesurvival of isolated stem cells by at least 2-fold compared to theabsence of the PKC activator.

DEFINITIONS

The abbreviations used herein have their conventional meaning within thechemical and biological arts.

The term “stabilizing a cell” refers to substantially reducing oreliminating the response of a cell to a change in the conditions orenvironment to which the cell is exposed. “Substantially reducing” inthis context means that the response is at least 50% less than whatwould have occurred in the absence of a stabilizing component (e.g., thecompounds of the invention).

The term “changing the conditions or environment of a cell” refers tochanging the temperature, culture media (e.g., carbon source, saltconcentration, growth factor), dissociating the cells into isolatedcells, thawing cells, or otherwise changing a factor of a cell'simmediate environment. As discussed herein, changing the condition orenvironment of a cell will often change the cell's phenotype or cellularprogramming. For example, stem cells, as well as some other cells, whenisolated will differentiate and/or die in response to certain changessuch as isolation, thawing, etc. Thus, changing conditions can reduce oreliminate cell viability whereas stabilized cells as described herein donot have substantially reduced viability under the same changes ofcondition. Change in cell programming can also be monitored as a cell'sresponse to a specific stimulus that is characteristic for a certaincell type and/or as by expression of one or a set of characteristicgenes or gene products. As a non-limiting example, human pluripotentstem cells are known to express at least some, and optionally all, ofthe markers from the following list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81,TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Suchexpression may change as a stem cell loses pluripotency or otherwisedifferentiates. A stabilized human pluripotent stem cell would maintainits characteristic expression pattern following a change in condition.

An “isolated” cell has been substantially separated or purified awayfrom other cells of an organism.

The term “dissociating” cells refers to a process of isolating cellsfrom other cells or from a surface (e.g., a culture plate surface). Forexample, cells can be dissociated from an animal or tissue by mechanicalor enzymatic methods. Alternatively, cells that aggregate in vitro canbe dissociated from each other. In yet another alternative, adherentcells are dissociated from a culture plate or other surface.Dissociation thus can involve breaking cell interactions withextracellular matrix (ECM) and substrates (e.g., culture surfaces) orbreaking the ECM between cells.

“Determining a phenotype of a cell” refers to assessing a quality orcharacteristic of the cell. Phenotypes can include, for example, celltype-characteristic gene expression, or gene expression patterns,response of the cell to a stimulus or environment, ability todifferentiate or de-differentiate, have a particular morphology, etc.

Where chemical substituent groups are specified by their conventionalchemical formulae, written from left to right, they equally encompassthe chemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e. unbranched) or branched chain,or combination thereof, which may be fully saturated, mono- orpolyunsaturated and can include di- and multivalent radicals, having thenumber of carbon atoms designated (i.e. C₁-C₁₀ means one to tencarbons). Examples of saturated hydrocarbon radicals include, but arenot limited to, groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. Preferred alkyl groups are C₁₋₆ alkylgroups.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl, as exemplified, but not limited,by —CH₂CH₂CH₂CH₂—. Typically, an alkyl (or alkylene) group will havefrom 1 to 24 carbon atoms, with those groups having 10 or fewer carbonatoms being exemplified in the present invention. A “lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generallyhaving eight or fewer carbon atoms. Preferred alkylene groups are C₁₋₆alkylene groups.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, P and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to two heteroatoms maybe consecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—. As described above, heteroalkylgroups, as used herein, include those groups that are attached to theremainder of the molecule through a heteroatom, such as —C(O)R′,—C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where “heteroalkyl” isrecited, followed by recitations of specific heteroalkyl groups, such as—NR′R″ or the like, it will be understood that the terms heteroalkyl and—NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specificheteroalkyl groups, such as —NR′R″ or the like. Preferred heteroalkylgroups are C₁₋₆ heteroalkyl groups.

As used herein, the term “heteroalkylene” refers to a heteroalkyl group,as defined above, linking at least two other groups. The two moietieslinked to the heteroalkylene can be linked to the same atom or differentatoms of the heteroalkylene. Preferred heteroalkylene groups are C₁₋₆heteroalkylene groups.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and“heterocycloalkylene” refer to a divalent radical derived fromcycloalkyl and heterocycloalkyl, respectively. Cycloalkyl andheterocycloalkyl groups can be C₃₋₈ cycloalkyl and C₃₋₈ heterocycloalkylgroups, or C₅₋₈ cycloalkyl and C₅₋₈ heterocycloalkyl groups

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a carbon or heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. “Arylene” and “heteroarylene”refers to a divalent radical derived from a aryl and heteroaryl,respectively. Aryl groups of the present invention preferably have 5-12ring members, more preferably 6-10 ring members. Heteroryl groups of thepresent invention preferably have 5-12 ring members, more preferably5-10 ring members.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

The term “oxo” as used herein means an oxygen that is double bonded to acarbon atom.

The term “alkylsulfonyl” as used herein means a moiety having theformula —S(O2)-R′, where R′ is an alkyl group as defined above. R′ mayhave a specified number of carbons (e.g. “C1-C4 alkylsulfonyl”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Exemplary substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₆)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′— or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C′″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″ and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   (A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted    alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,    unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted    heteroaryl, and-   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and    heteroaryl, substituted with at least one substituent selected from:    -   (i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted        alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl, substituted with at least one substituent selected        from:        -   (a) oxo, —OH, —NH₂, —SH, —CN, —CF, —NO₂, halogen,            unsubstituted alkyl, unsubstituted heteroalkyl,            unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,            unsubstituted aryl, unsubstituted heteroaryl, and        -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            or heteroaryl, substituted with at least one substituent            selected from oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,            unsubstituted alkyl, unsubstituted heteroalkyl,            unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,            unsubstituted aryl, and unsubstituted heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein meansa group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl isa substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

Thus, the compounds of the present invention may exist as salts withpharmaceutically acceptable acids. The present invention includes suchsalts. Examples of such salts include hydrochlorides, hydrobromides,sulfates, methanesulfonates, nitrates, maleates, acetates, citrates,fumarates, tartrates (eg (+)-tartrates, (−)-tartrates or mixturesthereof including racemic mixtures, succinates, benzoates and salts withamino acids such as glutamic acid. These salts may be prepared bymethods known to those skilled in the art.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivaenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,tautomers, geometric isomers and individual isomers are encompassedwithin the scope of the present invention. The compounds of the presentinvention do not include those which are known in the art to be toounstable to synthesize and/or isolate.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²¹I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areencompassed within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Novel synthetic small molecules dramatically increase hESCsurvival after single cell dissociation without compromising theirlong-term self-renewal and full developmental potential. (a) Chemicalstructures of Thiazovivin/Tzv and Pyrintegrin/Ptn as indicated. (b) ALPstaining of hESC colonies that had grown from dissociated single cellsseeded in low density and treated as indicated. (c) Ratio of ALPpositive colonies vs. total initially seeded cells. (d) Immunostainingof hESCs long-term maintained in media containing Ptn or Tzv asindicated. (e) Sections of 5 weeks teratomas formed from long-termexpanded hESCs maintained in media containing Tzv (i, ii) or Ptn (iii,iv). Neuroepithelium (ectoderm), cartilage (mesoderm), and simpleepithelium (endoderm) (i); neuroepithelium (ectoderm), simple epitheliumand hepatic-type epithelium (endoderm) (ii); neuroepithelium (ectoderm),cartilage (mesoderm) and tubular epithelium (endoderm) (iii);neuroepithelium (ectoderm), skeletal muscle (mesoderm), and tubularepithelium (endoderm) (iv). (f) G-banding analysis of hESCs after morethan 20 passages, propagated in the presence of compounds Ptn or Tzv. Ifnot specified, all the above hESCs were grown in the chemically-definedmedium and feeder-free on the Matrigel-coated plates.

FIG. 2. Tzv stabilizes E-cadherins after cell dissociation to protecthESCs from death in suspension culture. (a) Cell death analysis ofdissociated hESCs grown on Matrigel or in suspension treated with orwithout Ptn or Tzv. (b) Phase contrast images of hESCs grown onnon-coated plates treated with the indicated molecules. (c) Western blotanalysis of E-cadherins in hESCs that were transfected with the specificsiRNAs against E-cadherin or GFP. (d) Cell death analysis by TUNELstaining of and (e) ALP staining of dissociated hESCs that weretransfected with the specific siRNAs against E-cadherin or GFP in thepresence Tzv. (f) Western blot analysis of full-length E-cadherins inhESCs before and after trypsin. (g) A time-course Western blot analysisof full-length E-cadherin expression in hESCs after trypsin dissociationand treatment with DMSO, Tzv, or Ptn for indicated time. (h) Flowcytometry analysis of E-cadherin surface level in hESCs after trypsintreatment in the presence of Tzv. DMSO was used as a control. (i)Semiquantitative RT-PCR of E-cadherin in hESCs treated with or withoutTzv. (j) E-cadherin endocytosis analysis in the absence or presence ofTzv. (k) Cell survival analysis of hESCs grown on BSA- or differentconcentrations of E-cad-Fc chimera-coated plates.

FIG. 3. Ptn and Tzv protect hESCs from cell death in adherent cultureafter dissociation by maintaining and re-activating integrin activity.(a) Growth curve of hESCs grown on Matrigel with different time coursesof Ptn and Tzv treatment. Group 1, Ptn treatment during the first 24 honly; Group 2, continuous Ptn treatment during the entire cultureperiod; Group 3, Tzv treatment during the first 24 h only; Group 4,continuous Tzv treatment during the culture; For each condition, 10×10⁴dissociated cells were plated per well of a 6-well plate. (b) Phasecontrast images of hESCs 12 hours after seeding on the differentmatrices and treated with the indicated compounds. (c) Dissociated hESCswere plated on Matrigel-coated plates and allowed to adhere for 3 h inthe presence of compounds or together with integrin β1 blocking antibodyas indicated. The percentage of adhesion was calculated as described inthe Materials and Methods. (d) Western blot analysis of integrin β1expressed by hESCs before and after trypsin treatment. (e) A time coursewestern blot analysis of integrin expression in hESCs after trypsindissociation and treatment with DMSO, Tzv, or Ptn for indicated time.(f, g) Flow cytometry (f), and immunostaining (g) analysis of the β1integrins in the active conformation in trypsin-dissociated hESCs aftertreatment with Tzv or Ptn. (h, i) Cell adhesion (h) and ALP staining (i)of hESCs treated with or without β1-activating antibody, TS2/16. (j)Cell adhesion of hESCs treated with Tzv or Ptn in combination with orwithout a PKC inhibitor. (k) Immunostaining of β1 integrins in theactive conformation in hESCs treated with or without PMA (10 nM). (l, m)Cell adhesion (l) and ALP staining (m) of hESCs treated with theindicated compounds.

FIG. 4. Growth factors receptors-mediated PI-3 K and ERK are the majorsurviving and anti-differentiation signaling generated from the hESCniche, respectively. (a) Cell death analysis of dissociated hESCs platedon Matrigel and treated as indicated. (b) Western blot showing thephosphorylation status of different growth factor receptors in hESCstreated with Ptn for 2 h. DMSO was used as a control. (c) Cell deathanalysis of dissociated hESCs in suspension treated with the indicatedconditions. (d) Immunoprecipitation showing the interaction ofE-cadherins with EGFR1 and Erb2. (e) Western blot showing AKTphosphorylation status in hESCs treated with Ptn for the indicated timeperiods. (f) Western blot showing AKT and ERK phosphorylation status inthe presence of Ptn or together with integrin β1 blocking antibody. (g)Western blot showing AKT phosphorylation status in hESCs treated withPtn or together with the indicated receptor inhibitors. (h) Cell deathanalysis of hESCs treated with Ptn or together with PI-3 K inhibitor, orMEK inhibitor for 24 h. (i) Percentage of SSEA4 negative cells aftertreatment with MEK inhibitor.

FIGS. 5A and 5B show compounds of the present invention, includingthiazovivin and derivatives thereof.

FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J, 6K, 6L, 6M, 6N, 6O, 6P,6Q, 6R, 6S, 6T, 6U, 6V, 6W, 6X, 6Y, 6Z, 6AA, 6AB and 6AC show compoundsof the present invention, including pyrintegrin and derivatives thereof.

DETAILED DESCRIPTION I. Introduction

The present invention provides novel compounds as well as methods fortheir use. Two classes of small molecule chemical compounds are providedthat prevent differentiation of cells and promote cell survival,including but not limited to, when the cells are isolated or areotherwise outside their normal medium or tissue milieu. The compoundswork by somewhat different mechanisms but both are useful asprophylactic and therapeutic compounds for a number of different diseaseindications, including but not limited to, cancer, tissue damage, andstroke.

II. Compounds that Promote Cell Survival and/or Anti-Differentiation

In one aspect, compounds that promote cell survival and/oranti-differentiation are provided. In some embodiments, the compound hasthe formula:

In Formula (I), ring A is a substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. Ring Bis a substituted or unsubstituted heterocycloalkyl, or substituted orunsubstituted heteroaryl.

L¹ is —C(O)—NR²— or —NR²—C(O)—. L² is a bond, substituted orunsubstituted alkylene or substituted or unsubstituted heteroalkylene.

R¹ and R² are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In some embodiments, ring A is a substituted or unsubstituted aryl. RingA may also be a substituted or unsubstituted phenyl.

In other embodiments, ring B is a substituted or unsubstitutedheterocycloalkyl, or substituted or unsubstituted heteroaryl. Ring B mayalso be a substituted or unsubstituted heteroaryl. In still otherembodiments, ring B is a substituted or unsubstituted pyrazolyl,substituted or unsubstituted furanyl, substituted or unsubstitutedimidazolyl, substituted or unsubstituted isoxazolyl, substituted orunsubstituted oxadiazolyl, substituted or unsubstituted oxazolyl,substituted or unsubstituted pyrrolyl, substituted or unsubstitutedpyridyl, substituted or unsubstituted pyrimidyl, substituted orunsubstituted pyridazinyl, substituted or unsubstituted thiazolyl,substituted or unsubstituted triazolyl, substituted or unsubstitutedthienyl, substituted or unsubstituted dihydrothieno-pyrazolyl,substituted or unsubstituted thianaphthenyl, substituted orunsubstituted carbazolyl, substituted or unsubstituted benzothienyl,substituted or unsubstituted benzofuranyl, substituted or unsubstitutedindolyl, substituted or unsubstituted quinolinyl, substituted orunsubstituted benzotriazolyl, substituted or unsubstitutedbenzothiazolyl, substituted or unsubstituted benzooxazolyl, substitutedor unsubstituted benzimidazolyl, substituted or unsubstitutedisoquinolinyl, substituted or unsubstituted isoindolyl, substituted orunsubstituted acridinyl, substituted or unsubstituted benzoisazolyl, orsubstituted or unsubstituted dimethylhydantoin.

L² may be substituted or unsubstituted C₁-C₁₀ alkyl. In someembodiments, L² is unsubstituted C₁-C₁₀ alkyl. L² may also besubstituted or unsubstituted methylene (e.g. unsubstituted methylene).

R² may be hydrogen. R¹ may be hydrogen or unsubstituted C₁-C₁₀ alkyl. Insome embodiments, R¹ is simply hydrogen.

In some embodiments of Formula (I), ring A is substituted orunsubstituted aryl, ring B is substituted or unsubstituted heteroaryl,R¹ is hydrogen, and L² is unsubstituted C₁-C₁₀ alkyl.

In another embodiment, the compound that promote cell survival and/oranti-differentiation has the formula:

In Formula (II), y is an integer from 0 to 3 and z is an integer from 0to 5. X is —N═, —CH═ or —CR⁵═. R¹ and L² are as defined above in thedefinitions of Formula (I).

R³, R⁴ and R⁵ are independently —CN, —S(O)_(n)NR⁶, —NR⁷R⁸, —C(O)R⁹,—NR¹⁰—C(O)R¹¹, —NR¹²—C(O)—OR¹³, —C(O)NR¹⁴R¹⁵, —NR¹⁶S(O)₂R¹⁷, —OR¹⁸,—S(O)₂NR¹⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, whereinn is an integer from 0 to 2, wherein if z is greater than 1, two R³moieties are optionally joined together to form a substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸ and R¹⁹ areindependently hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In some embodiments, L² is substituted or unsubstituted C₁-C₁₀ alkyl. L²may also be unsubstituted C₁-C₁₀ alkyl. Alternatively, L² is substitutedor unsubstituted methylene (e.g. unsubstituted methylene).

In other embodiments, X is —N═ or —CH═. The symbol z may be 2. In stillother embodiments, two R³ moieties at adjacent vertices are joinedtogether to from a substituted or unsubstituted heterocycloalkyl. Thesymbol z may also be 1. The symbol y may be 0 or 1. R³ may be —OR¹⁸. R¹⁸may be hydrogen or unsubstituted C₁-C₁₀ alkyl.

In some embodiments, L² is substituted or unsubstituted methylene (e.g.substituted methylene), X is —N═ or —CH═, R¹ is hydrogen, and y and zare 0.

In other embodiments, the compounds has the formula:

In still other embodiments, the compounds of formula I are those wherering A is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, eachoptionally substituted with 1-5 R³ groups; ring B is heterocycloalkyl orheteroaryl, each optionally substituted with 1-5 R⁴ groups; L¹ is—C(O)—NR²— or —NR²—C(O)—; L² is a bond, C₁₋₁₀ alkylene or C₁₋₁₀heteroalkylene; R¹ and R² are independently hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀heteroalkyl, C₃₋₈ cycloalkyl, C₃₋₈ heterocycloalkyl, aryl, orheteroaryl; each R³ and R⁴ is independently —CN, —S(O)_(n)R⁶, —NR⁷R⁸,—C(O)R⁹, —NR¹⁰—C(O)R¹¹, —NR¹²—C(O)—OR¹³, —C(O)NR¹⁴R¹⁵, —NR¹⁶S(O)₂R¹⁷,—OR¹⁸, —S(O)₂NR¹⁹, C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl, wherein n is an integer from 0 to2, wherein two R³ moieties are optionally joined together to form acycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are eachindependently hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl. In still yet other embodiments,the compounds of formula I are other than Thiazovivin.

In other embodiments, the compound that promote cell survival and/oranti-differentiation has the formula:

In Formula (III), ring D is substituted or unsubstituted aryl orsubstituted or unsubstituted heteroaryl. L³ is —C(O)NH— or —S(O)₂NH—.

R²⁰ is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R²¹ is —NR²²R²³ or —OR²⁴.

R²² and R²³ are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, or joined together to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl.

R²⁴ is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or joined together to form asubstituted or unsubstituted cycloalkyl of substituted or unsubstitutedheterocycloalkyl.

In other embodiments, L³ is a bond, —O—, —C(O)NH— or —S(O)₂NH—, R²⁰ ishydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, and RingD and R²¹ are as defined above. In some other embodiments, L³ is a bond,—O— or —S(O)₂NH—, and Ring D, R²⁰ and R²¹ are as defined above, suchthat when L³ is —S(O)₂NH—, R²⁰ is hydrogen. In still other embodiments,L³ is a bond or —O—, and Ring D, R²⁰ and R²¹ are as defined above.

In some embodiments, ring D is substituted or unsubstituted phenyl.

In other embodiments, R²⁰ is substituted or unsubstituted alkyl orsubstituted or unsubstituted cycloalkyl. R²⁰ may be substituted orunsubstituted C₁-C₁₀ alkyl or substituted or unsubstituted 3 to 7membered cycloalkyl. R²⁰ may also be substituted or unsubstituted C₁-C₅alkyl or substituted or unsubstituted 3 to 6 membered cycloalkyl. Insome embodiments, R²⁰ is unsubstituted C₁-C₅ alkyl or unsubstituted 3 to6 membered cycloalkyl.

In still other embodiments, R²² is hydrogen, and R²³ is substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. Alternatively, R²² is hydrogen; and R²³ is substituted orunsubstituted substituted or unsubstituted aryl. Or R²² and R²³ arejoined together to from a substituted or unsubstituted heterocycloalkyl,or substituted or unsubstituted heteroaryl. R²² and R²³ may also bejoined together to from a substituted or unsubstituted pyrrolyl,substituted or unsubstituted isoindolinyl, substituted or unsubstitutedpiperidinyl, or substituted or unsubstituted tetrahydroquinolinyl.

In some embodiments, the compound has the formula:

In Formula (IV), w is an integer from 0 to 1 and q is an integer from 0to 7. R²⁰ is as defined above in the definition of the compound ofFormula (III). R²⁵, R²⁶, R²⁷ and R²⁸ are independently —CN, —NR²⁹R³⁰,—C(O)R³¹, —NR³²—C(O)R³³, —NR³⁴—C(O)—OR³⁵, —C(O)NR³⁶R³⁷, —NR³⁸S(O)₂R³⁹,—OR⁴⁰, —S(O)₂NR⁴¹, —S(O)_(v)R⁴², substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl,wherein v is an integer from 0 to 2.

R²⁹, R³⁰, R³¹, R³², R³³, R³⁴, R³⁵, R³⁶, R³⁷, R³⁸, R³⁹, R⁴⁰, R⁴¹, and R⁴²are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R²⁵ and R²⁶, or R²⁶ and R²⁷, may be joined to form a substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In some embodiments, R²⁸ is —OR⁴⁰. R⁴⁰ is hydrogen or unsubstitutedC₁-C₁₀ alkyl R⁴⁰ may also be hydrogen or unsubstituted C₁ to C₅ alkyl.

The compound may also have the formula:

In Formula (V), R²⁰, R²⁸, and q are as defined above in the definitionsof Formula (IV). In some embodiments, R²⁰ is unsubstituted C₁ to C₁₀alkyl. R²⁸ may be —OR⁴⁰. R⁴⁰ is hydrogen or unsubstituted C₁ to C₁₀alkyl, or C₁ to C₁₀ alkyl substituted with substituted or unsubstitutedC₃ to C₆ cycloalkyl. The symbol q may be 1.

In another embodiment, the compound has the formula:

In Formula (VI), R²⁰, R²⁸, and q are as defined above in the definitionsof Formula (IV) or Formula (VI).

In another embodiment, the compound has the formula:

In other embodiments, the compounds of formula III are those where RingD is aryl or heteroaryl, each optionally substituted with 1-5 R groups;L³ is —C(O)NH— or —S(O)₂NH—; R²⁰ is C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionallysubstituted with 1-5 R groups; R²¹ is —NR²²R²³ or —OR²⁴; R²² and R²³ areindependently hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, or are joined together to form aheterocycloalkyl or heteroaryl, each optionally substituted with 1-5 Rgroups; R²⁴ is C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, each optionally substituted with 1-5R groups; and each R group is independently selected from the groupconsisting of C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN, —NO₂, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl,wherein each R′, R″, R′″ and R″″ is independently selected from thegroup consisting of hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, and arylalkyl groups.

In some embodiments, each substituted group described above in thecompounds of Formulae (I)-(VI) is substituted with at least onesubstituent group. More specifically, in some embodiments, eachsubstituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, substituted aryl, substituted heteroaryl,substituted alkylene, and/or substituted heteroalkylene described abovein the compounds of Formulae (I)-(VI) are substituted with at least onesubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one size-limited substituent group.Alternatively, at least one or all of these groups are substituted withat least one lower substituent group.

In other embodiments of the compounds of Formulae (I)-(VI), eachsubstituted or unsubstituted alkyl is a substituted or unsubstitutedC₁-C₂₀ alkyl, each substituted or unsubstituted heteroalkyl is asubstituted or unsubstituted 2 to 20 membered heteroalkyl, eachsubstituted or unsubstituted cycloalkyl is a substituted orunsubstituted C₃-C₈ cycloalkyl, each substituted or unsubstitutedheterocycloalkyl is a substituted or unsubstituted 3 to 8 memberedheterocycloalkyl, each substituted or unsubstituted alkylene is asubstituted or unsubstituted C₁-C₂₀ alkylene, and/or each substituted orunsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20membered heteroalkylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl, and/or each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₈ alkylene, and/or eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene.

In some other embodiments, the compounds of formula (I)-(VI) can besubstituted with C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR′″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN, —NO₂, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl,wherein each R′, R″, R′″ and R″″ hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or arylalkyl groups.

III. Methods of Use

The compounds of the present invention are useful for a wide variety ofpurposes. For example, the compounds promote survival in situations(e.g., for isolated cells) where the cells would otherwise go throughapoptosis or otherwise die. In some embodiments, the cells arestabilized for at least a particular period of time, e.g., 10 minutes,30 minutes, or 1, 2, 4, 6, 8, 10, 24, 48, or 96 hours. Further, thecompounds are useful in maintaining the current state of differentiationof cells in conditions where the cells would otherwise differentiate orotherwise change their programming. These effects lead to a large numberof uses for the compounds either in vitro or in vivo.

A. In Vivo Uses

The compounds of the invention are useful for reducing tissue damage andthus can be administered to treat, ameliorate, or prevent tissue damage.In some embodiments, a compound of the invention is administered to anindividual having, or at risk of having tissue damage to an internalorgan. Internal organs include, but are not limited to, brain, pancreas,liver, intestine, lung, kidney, or heart, wounding, e.g., by burn orcut. For example, in some embodiments, the compounds of the inventionare effective in reducing infarction size in reperfusion followingischemia. Thus, a compound of the invention can be administered toindividuals at risk of having, having, or who have had, a stroke.Similarly, a compound of the invention can be administered toindividuals at risk of having, having, or who have had, a heart attackor cardiac damage.

The inventors have found that the compounds of the invention can preventcell death, for example in epithelial cells. For example, the inventorsdispersed primary human pancreatic islets/beta cells plated as singlecells onto a tissue culture plate that was coated with matrigel orlaminin. In regular cell culture media for beta cells without Tzvresulted in substantial cell death. However, when Tzv was added to themedia (1-2 mM), cell death was inhibited. The same effect was observedfor other epithelial primary cells, such as neural cells. Accordingly,in some embodiments, a compound of the present invention is administeredto an individual in need of pancreatic beta and/or islet cells, whereinadministration of the compound results in an increase in the number ofbeta or islet cells in the individual.

Further, the compounds of the invention (e.g., those of Formulae I orIII) are effective in increasing blood flow and inhibiting inflammatoryresponses. For example, compounds of Formula I enhance adhesion andmigration of monocytes across monolayers of endothelial cells and canthus relieve inflammatory responses (data not shown). Thus, in someembodiments, a compound of the invention is administered to anindividual (e.g., having cerebral ischemia) in need of increased bloodflow and/or decreased inflammation. Those in need of reducedinflammation include individuals with inflammatory disease or with adisease mediated by an inflammatory condition. Exemplary inflammatorydiseases include, but are not limited to, chronic obstructive pulmonarydisease, osteoarthritis, tendinitis or bursitis, gouty arthritis,polymyalgia rheumatica, fibromyalgia, pelvic inflammatory disease andarthritis, including rheumatoid arthritis

In some embodiments, the compounds of the present invention are used totreat or ameliorate cancer. In some cases, a compound of the presentinvention is administered to treat cancer, e.g., carcinomas, gliomas,mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas, breastcancer, ovarian cancer, cervical cancer, glioblastoma, leukemia,lymphoma, prostate cancer, and Burkitt's lymphoma, head and neck cancer,colon cancer, colorectal cancer, non-small cell lung cancer, small celllung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer,hepatobiliary cancer, cancer of the gallbladder, cancer of the smallintestine, rectal cancer, kidney cancer, bladder cancer, prostatecancer, penile cancer, urethral cancer, testicular cancer, cervicalcancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer,parathyroid cancer, adrenal cancer, pancreatic endocrine cancer,carcinoid cancer, bone cancer, skin cancer, retinoblastomas, Hodgkin'slymphoma, non-Hodgkin's lymphoma (see, CANCER: PRINCPLES AND PRACTICE(DeVita, V. T. et al. eds 1997) for additional cancers).

Cancer cell metastasis is typically a process involving epithelial tomesenchymal transition/EMT (e.g. from epithelial-type cells tofibroblast-type cells). The inventors have found that the compounds ofthe invention (i.e., Tvz and Ptn) can induce MET (the reverse of EMT)and inhibit EMT, indicating that compounds are effective at reducing orpreventing cancer metastasis. Accordingly, in some embodiments, acompound of the present invention is administered to an individualhaving or at risk of having cancer metastasis. For example, theinventors have found that compounds of formula I and III inhibitmetastasis of epithelial cancers including but not limited to breastcancer and hepatocellular carcinoma.

In some embodiments, a compound of the present invention is administeredto an individual having, or at risk of having, hypertension and/oratherosclerosis.

Compounds of formula I and III (i.e., Tvz and Ptn) are effective atpromoting axonal regeneration and functional recovery in injured centralnervous system. For example, the inventors have found that Tvz canpromote neurite outgrowth from primary neuronal cells from mice. Tzv (3μM) was tested on mouse PI cortical explants, with axon outgrowth as anoutcome read-out. Tzv was added to the medium 20 minutes after plating,with DMSO as a control. The explants were observed for 4 days inculture. Tzv showed a dramatic effect in promoting axon outgrowth, whichwas notable from 1 div. Accordingly, in some embodiments, a compound ofthe present invention is administered to an individual having a centralnervous system injury or who is in need or would otherwise benefit fromaxonal regeneration.

In some embodiments, a compound of the present invention is administeredto an individual having diabetes, insulin resistance, or otherwise inneed to promotion of beta cell survival, or at risk of having loss ofbeta cell function.

The compounds of the invention also find use in ameliorating negativesymptoms of, or otherwise improving, organ, cell, or tissuetransplantation. As explained herein, the compounds of the invention areeffective in stabilizing and maintaining contextual programming ofcells. Thus, in some embodiments, a compound of the invention isadministered during and after transplantation of cells, tissue or anorgan to an individual. Examples of transplantation include, but are notlimited to, transplantation of bone marrow, umbilical cord blood,purified hematopoietic stem/progenitor cells, cardiac cells, neuralcells, pancreatic beta cells, and liver cells.

B. In Vitro Uses

The compounds of the present invention are effective at stabilizingcells exposed to a wide variety of conditions. Many animal cells, whenisolated (in suspension or alternatively, when adherent) lose viability,go through apoptosis, and/or change programming (for example, stem cellswhen isolated, will often die or differentiate). The compounds of thepresent invention, when mixed with such cells, are effective inpreventing such cellular responses to environmental changes. In someembodiments, cells are isolated from an animal and contacted with acompound of the invention in a sufficient amount to prevent loss of cellviability and/or changes in cellular programming. In some embodiments,such isolated cells are useful for diagnostics as the cells isolatedretain phenotypes that would otherwise be lost due to the cell'sresponse to the isolation process and isolation itself. Exemplaryretained phenotypes can include, for example, gene expression patterns,cell responsiveness to a stimulus, ligand, or drug, cell viability.

Stability of a cell population can be monitored, for example, bymonitoring expression of gene products. For example, certain geneproducts are tissue or cell type-specific and can be monitored beforeand after a change in condition or environment (for example, changing ofcell media, thawing of cell, isolation of cell from other cells, etc.)to determine whether the change affects cellular programming. In someembodiments, cells about to be submitted to a change of condition orenvironment, or relatively soon after (e.g., within 1 minute, 5 minutes,one hour, etc., depending on circumstances) the change, are contactedwith a compound of the invention in a sufficient amount such that one ormore cellular expression markers remain substantially the same.“Substantially the same” will depend upon context and will be understoodin the art. In some embodiments, “substantially the same” means thatexpression of a gene product associated with a specific cell type doesnot change more than about 10%, 20% or 30% following a particulartreatment to the cell (e.g., compared to expression prior to thetreatment).

In some embodiments, the invention provides methods of promotingsurvival and anti-differentiation in stem cells ex-vivo. For example,the inventors have found that compounds of Formulae I or III (i.e., Tzvand Ptn) are effective in promoting survival and anti-differentiation inhuman embryonic stem cell, mouse embryonic stem cell, multiple neuralstem cells, skin stem cells, mesenchymal stem cells, hematopoietic stemcells, stromal stem cells and epithelial stem cells by contacting thecells with a compound of Formula I or III immediately after theisolation of the cells

Accordingly, the present invention provides populations of cells and/ortissue in contact with a sufficient amount of a compound of theinvention (e.g., a compound of Formula I or III) to stabilize the cells,e.g., to prevent or reduce cellular responses to changes in conditions(e.g., isolation from a tissue, thawing of the cells, etc.). In someembodiments, for example, the cells or tissues in contact with acompound of the invention are in a frozen or a liquid states. In someembodiments, the cells/tissues are thawed from a frozen state while incontact with a sufficient amount of a compound of the invention toprevent or reduce cellular damage or differentiation.

In some embodiments, a compound of the invention is contacted to apopulation of stem cells, progenitor cells or differentiated cells.Exemplary stem cells include pluripotent stem cells, embryonic stemcells, induced stem cells (iPS cells). Exemplary stem cells also includehuman embryonic stem cells, mouse embryonic stem cells, multiple neuralstem cells, skin stem cells, mesenchymal stem cells, hematopoietic stemcells, stromal stem cells, and epithelial stem cells. Any type ofprogenitor cells can be used, including but not limited to, endodermprogenitor cells, mesoderm progenitor cells (e.g., muscle progenitorcells, bone progenitor cells, blood progenitor cells), and ectodermprogenitor cells (e.g., epidermal tissue progenitor cells and neuralprogenitor cells). There are a wide variety of differentiated cellsknown. Differentiated cells include, but are not limited to,fibroblasts, cardiac cells, neural cells, pancreatic beta cells, livercells, epithelial cells, and intestinal cells. The cells describedherein can be human cells or non-human cells. In some embodiments, thecells are human cells. In some embodiments, the cells are mouse, dog,cow, pig, rat or non-human primate cells.

The ability to maintain cell viability and cellular programming allowfor improved methods of drug screening and diagnostics. For example, insome embodiments, a cell is screened for a response in the presence ofat least one compound of the invention (e.g., a compound of formula I orIII), thereby maintaining viability of the cell, and further contactedwith at least one of a plurality of agents (e.g., a chemical library)and then monitored for a response. A wide range of screening methods areknown. This method finds particular benefit for use with cells thatwould otherwise have a poor viability in the conditions of the screeningmethod (for example, where it is convenient to use isolated cells, cellsin suspension, adhesive cells, etc.). The cells can be, for example,stem cells, progenitor cells or differentiated cells, as describedherein. The cellular response can be any response desired. Someresponses in cell-based screening assays include, but are not limitedto, expression of a gene (e.g., based on expression of a reporter geneor quantified by PCR or other detection technology), cell viability orloss thereof, induction of apoptosis, etc.

Agents used in the screening methods can be, for example, small organiccompounds (e.g., molecular weight less than 10,000 daltons, for example,less than 8000, 6000, 4000, 2000 daltons), lipids, sugars, polypeptides,antibodies, nucleic acids (e.g., oligonucleotides, DNA, RNA, ribozymes,short inhibitory RNA (siRNA), micro RNA (miRNA), etc.).

In some embodiments, the assays are designed to screen largecombinatorial libraries by automating the assay steps and providingcompounds from any convenient source to assays, which are typically runin parallel (e.g., in microtiter formats or in microwell plates inrobotic assays). The combinatorial libraries can be completely random,or comprise members that contain a core structure based on one or morepromising lead compounds. The combinatorial libraries can be completelysynthetic or can include some or all members that are derived fromnaturally occurring sources, including, for example, bacteria, fungi,plants, insects and vertebrate (e.g., Xenopus (frog) or Anguilla (eel))and non-vertebrate animals (e.g., Strongylocentrotus (sea urchin) ormollusks). See also, Boldi, Combinatorial Synthesis of Natural ProductBased Libraries, 2006, CRC Press.

In one embodiment, high throughput screening methods involve providing acombinatorial chemical or peptide library containing a large number ofpotential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. See, for example, U.S. Pat. Nos.5,663,046; 5,958,792; 6,185,506; 6,541,211; 6,721,665, the disclosuresof which are hereby incorporated herein by reference. Such combinatorialchemical libraries include, but are not limited to, peptide libraries(see, e.g., U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res.37:487-493 (1991); Houghton, et al., Nature 354:84-88 (1991); andCombinatorial Peptide Library Protocols, Cabilly, ed., 1997, HumanaPress. Other chemistries for generating chemical diversity libraries canalso be used. Such chemistries include, but are not limited to: peptoids(e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCTPublication WO 93/20242), random bio-oligomers (e.g., PCT PublicationNo. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514),diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs etal., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogouspolypeptides (Hagihara et al, J. Amer. Chem. Soc. 114:6568 (1992)),nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al.,J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic synthesesof small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661(1994); Combinatorial Libraries: Synthesis, Screening and ApplicationPotential, Cortese, ed., 1995, Walter De Gruyter Inc; and Obrecht andVillalgordo, Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compound Libraries, 1998, Elsevier Science Ltd),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, infra, Sambrook and Russell, infra and U.S.Pat. Nos. 6,955,879; 6,841,347; 6,830,890; 6,828,098; 6,573,098; and6,399,334), peptide nucleic acid libraries (see, e.g., U.S. Pat. Nos.5,539,083; 5,864,010 and 6,756,199), antibody libraries (see, e.g.,Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,Science, 274:1520-1522 (1996); U.S. Pat. No. 5,593,853; and SolidSupport Oligosaccharide Synthesis and Combinatorial CarbohydrateLibraries, Sceberger, ed., 2004, John Wiley & Sons (E-book)), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993) and U.S. Pat. No. 5,288,514; isoprenoids,U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat.No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;morpholino compounds, U.S. Pat. No. 5,506,337, and the like). See also,Combinatorial Library Design and Evaluation: Principles, Software Tools,and Applications in Drug Discovery, Ghose, et al., eds., 2001, MarcelDekker; Molecular Diversity and Combinatorial Chemistry: Libraries andDrug Discovery, Chaiken and Janda, eds., 1996, Oxford Univ Pr.; andCombinatorial Library Method and Protocols, English, ed., 2002, HumanaPress.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., Advanced Chem Tech, Louisville Ky., Symphony,Rainin, Woburn, Mass., Applied Biosystems, Foster City, Calif.,Millipore, Bedford, Mass. and Caliper Life Sciences, Hopkinton, Mass.).

In some embodiments, the screening assays can be conveniently carriedout in multiwell plates (e.g., 96-well, 384-well, etc.) wherein eachagent to be screened is individually tested in a single well. In someembodiments, two or more candidate agents are tested in a singlereaction mixture.

C. Alternative Targets for Obtaining Similar Effects

As described in detail in the examples below, the inventors have learnedabout the role of several gene products in the cellular response to thecompounds of the invention and this has lead to the discovery that cellscan also be stabilized by manipulating the gene products as explainedbelow.

1. E-Cadherin

The inventors have found that increasing expression of E-cadherinenhances stem cell survival. Thus, the present invention provides formethods of stabilizing and/or increasing expression of E-cadherin in acell, thereby stabilizing the cell from a change of conditions thatwould otherwise be detrimental to viability of the cell. StabilizingE-cadherin can include, for example, contacting the cells with acompound that increases expression of E-cadherin or in some way protectsE-cadherin from proteolytic cleavage.

In some embodiments, the invention provides for methods of culturingstem cells (including but not limited to, human or mouse embryonic stemcells) in a container having a surface coated with a protein comprisingat least an ectodomain of E-cadherin, optionally linked to anothercomponent such as a fusion protein, thereby stabilizing the cells (e.g.,maintaining or increasing viability of the cells and/or maintainingcellular programming). An ectodomain is the part of a membrane proteinthat extends into the extracellular space (the space outside a cell). Insome embodiments, ectodomains are the part of a protein that initiatecontact with surface which leads to signal transduction. The ectodomainof E-cadherin is described in, e.g., Ito et al., Oncogene 18(50):7080-90(1999) In some embodiments, at least the ectodomain of E-cadherin isfused to a dimerizing polypeptide sequence, thereby allowing forstabilized dimers of the ectodomain. A “dimerizing polypeptide” refersto an amino acid sequence that forms homo-dimers, thereby allowing twopolypeptides to dimerize. Exemplary dimerizing polypeptides include, butare not limited to, an IgG Fc fragment. In some embodiments, in someembodiments, stem cells (including but not limited to human or mouseembryonic stem cells, pluripotent stem cells, iPS cells) are dissociatedfrom each other and cultured in a container having a surface coated witha protein comprising at least an ectodomain of E-cadherin, optionallylinked to another component such as a fusion protein, therebystabilizing the cells in improving the survival rate of the cellscompared to the survival rate of similarly treated cells cultured in acontainer lacking the polypeptide coating.

2. Protein Kinase C

The present invention also provides for stabilizing cells by contactingthe cells with a protein kinase C activator. As explained herein,treatment of dissociated hESCs with a protein kinase C activator grownin the presence of a matrigel matrix resulted in significantly improvedcell adhesion and colony formation, thereby improving cell viability.Accordingly, the invention provides for improving cell viability byculturing cells in the presence of a protein kinase C activator, therebyimproving cell viability, growth, and/or adhesion. In some embodiments,a protein kinase C activator is contacted to a populations of stemcells, progenitor cells or differentiated cells in an amount sufficientto improve cell viability and/or survival and/or adhesion. Exemplarystem cells include pluripotent stem cells, embryonic stem cells, inducedstem cells (iPS cells) or as otherwise described herein. Exemplaryprotein kinase C activators include, but are not limited to, phorbolesters (e.g., phorbol 12-myristate 13-acetate (PMA) or phorbol esters asdescribed in US Patent Publication No. 20080226589) or peptide agonists(e.g., as described in U.S. Pat. No. 6,165,977).

3. Integrin β1

The present invention also provides for stabilizing cells by contactingthe cells with an integrin β1 activator. As explained herein, treatmentof dissociated hESCs with an integrin β1 activator, where the cells aregrown on lamin resulted in significantly improved cell adhesion andcolony formation, thereby improving cell viability. Accordingly, theinvention provides for improving cell viability by culturing cells inthe presence of an integrin β1 activator, thereby improving cellviability, growth, and/or adhesion. In some embodiments, an integrin β1activator is contacted to a populations of stem cells, progenitor cellsor differentiated cells in an amount sufficient to improve cellviability and/or survival and/or adhesion. Exemplary stem cells includepluripotent stem cells, embryonic stem cells, induced stem cells (iPScells) or as otherwise described herein. Exemplary an integrin β1activators include, but are not limited to an integrin β1 activatingantibody such as, e.g., TS2/16 (commercially available from, e.g.,Thermo Scientific. Rockfield, Ill.).

IV. Cell Populations

As discussed herein, the present invention provides for cells in amixture (e.g., a cell culture) with one or more compound as describedherein (e.g., a compound of formula I or III—including but not limitedto Tzv and Pt—or a protein kinase C activator or an integrin β1activator). In some embodiments, the compound is in the mixture at aconcentration sufficient to maintain viability or cellular programmingin response to a change of cellular environment or condition (e.g.,thawing). For example, in some embodiments, the compounds are in aconcentration of at least 0.1 nM, e.g., at least 1, 10, 100, 1000,10000, or 100000 nM, e.g., between 0.1 nM and 100000 nM, e.g., between 1nM and 10000 nM, e.g., between 10 nM and 10000 nM, e.g., between 1-10μM. In some embodiments, the mixtures are in a synthetic vessel (e.g., atest tube, Petri dish, etc.). Thus, in some embodiments, the cells areisolated cells (not part of an animal). In some embodiments, the cellsare adherent cells or cells in suspension. In some embodiments, thecells are isolated or dissociated from a tissue sample (e.g., a biopsy)from an animal (human or non-human), placed into a vessel, and contactedwith one or more compound as described herein (e.g., a compound ofFormula I or III). The cells can be subsequently cultured andoptionally, inserted back into the same or a different animal,optionally after the cells have been stimulated to differentiate into aparticular cell type or lineage, or following introduction of arecombinant expression cassette into the cells.

V. Culturing of Cells

Cells can be cultured according to any method known in the art. Cellscan be cultured in suspension or as adherent cells as appropriate.

In some embodiments, the cells (e.g., stem cells) are cultured incontact with feeder cells. Exemplary feeder cells include, but are notlimited to fibroblast cells, e.g., mouse embryonic fibroblast (MEF)cells. Methods of culturing cells on feeder cells are known in the art.

In some embodiments, the cells are cultured in the absence of feedercells. Cells, for example, can be attached directly to a solid culturesurface (e.g., a culture plate), e.g., via a molecular tether. Exemplarymolecular tethers include, but are not limited to, matrigel, anextracellular matrix (ECM), ECM analogs, laminin, fibronectin, orcollagen. Those of skill in the art however will recognize that this isa non-limiting list and that other molecules can be used to attach cellsto a solid surface. Methods for initial attachment of the tethers to thesolid surface are known in the art.

VI. Formulations and Methods of Administration

Formulations (e.g., comprising a compound of the present invention,including but not limited to suitable for administration include aqueousand non-aqueous solutions, isotonic sterile solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic, and aqueous and non-aqueous sterile suspensionsthat can include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. In the practice of this invention,compositions can be administered, for example, orally, nasally,topically, intravenously, intraperitoneally, or intrathecally. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials. Solutions and suspensionscan be prepared from sterile powders, granules, and tablets of the kindpreviously described. The modulators can also be administered as part ofa prepared food or drug.

The dose administered to a patient, in the context of the presentinvention, should be sufficient to induce a beneficial response in thesubject over time. The optimal dose level for any patient will depend ona variety of factors including the efficacy of the specific modulatoremployed, the age, body weight, physical activity, and diet of thepatient, on a possible combination with other drugs, and on the severityof the disease or condition in question. The size of the dose also willbe determined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular compoundor vector in a particular subject.

In determining the effective amount of an active ingredient to beadministered a physician may evaluate circulating plasma levels of thecompound or agent, compound or agent toxicity, and the production ofanti-compound or agent antibodies. In general, the dose equivalent of acompound or agent is from about 1 ng/kg to 10 mg/kg for a typicalsubject.

VII. Examples

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

To improve chemically-defined medium conditions and uncover themolecular mechanism of hESC death after single cell dissociation, weperformed a high throughput phenotypic screen of 50,000 syntheticcompounds to identify small molecules that promote hESC survival aftertrypsin dissociation. From the screen, two chemical classes wereidentified that significantly increased the cell survival afterdissociation and also maintained hESC colony morphology and alkalinephosphatase (ALP) expression. Further chemical optimizations andactivity analysis resulted in the discovery of two lead molecules, a2,4-disubstituted thiazole (named as Thiazovivin/Tzv) and a2,4-disubstituted pyrimidine (named as Pyrintegin/Ptn) (FIG. 1a ), forfurther functional and mechanistic characterizations.

TABLE 1 Activity Data Activity (% ALP positive colony Compound formationby hESCs)¹

24.1

4.5

5.2

20.3

5

23.5

23.2

8

2.9

5.7

3.1

6.2

6.1

3.5

3.6

3.2

3

3.4

3.1

5.2

3.1

3.3

3.3

3.1 ¹Ratio of ALP positive colonies vs. total initially seeded cells.

Compound Tzv or Ptn enhances the survival of single hESCs more than20-fold on Matrigel-coated plate after enzymatic dissociation (FIG. 1b,c ). hESCs had been serially passaged in Tzv or Ptn-containingchemically-defined medium for more than 20 generations. Under suchconditions, the cells homogenously maintained the characteristicmorphology of hESCs, the expression of typical pluripotency markers, andnormal karyotype (FIG. 1d, e ). When these cells were injected into nudemice, they generated complex teratomas consisting of all three primarygerm layer tissues (FIG. 1f ). These results, confirmed with severalindependent hESC lines, collectively and convincingly demonstrated thatboth compounds could substantially promote hESC survival withoutcompromise to self-renewal and full developmental potency.

hESCs are known to be difficult in forming embryoid bodies (EBs) insuspension culture after single cell dissociation due to extensive celldeath. Thus, we also tested whether Tzv or Ptn could promote survival ofdissociated hESCs in suspension. Interestingly, Tzv greatly improvedsurvival of hESCs in both adherent and suspension cultures. In contrast,Ptn only promoted survival of hESCs in adherent culture (e.g. onMatrigel-coated plate), but had no effect on suspension culture (FIG. 2a). These observations suggest that at least two distinct mechanisms areinvolved in these two types of cell death under ECM/Matrigel orsuspension conditions, and that Tzv and Ptn function differently. hESCsformed nice cell aggregates when grown in suspension and in the presenceof Tzv (FIG. 2b ), and could differentiate into various lineages (datanot shown). Because cell aggregation is most often mediated throughcell-cell adhesions and E-cadherin is the primary cell-cell adhesionmolecule, as well as highly expressed in hESCs (Eastham, A. M. et al.,Cancer Res 67 (23):11254-11262 (2007)), we tested the effect of aspecific E-cadherin blocking antibody on multicellular aggregateformation. When the cells were cultured in the presence of the antibody,the cell survival and formation of large, compact aggregates induced byTzv treatment was severely inhibited, indicating that the cell survivaland assembly of multicellular aggregates induced by Tzv involvefunctional E-cadherin (FIG. 2b ). In addition, knock-down of E-cadherinby specific siRNAs in hESCs dramatically reduced cell survival inducedby Tzv treatment, and significantly decreased the number of ALP positivecolonies (FIG. 2 c,d,e). These results suggest that Tzv enhances hESCsurvival in suspension, presumably acting through E-cadherin-mediatedcell-cell adhesion.

We then examined E-cadherin expression in hESCs after trypsindissociation. We found that most of the full length E-cadherin had beencleaved after trypsin dissociation (FIG. 2f ). This observation wasconsistent with the report that the extracellular region of E-cadherinhas an endoproteolytic cleavage site near the transmembrane domain(Damsky, C. H. et al., Cell 34 (2):455-466 (1983)). In Tzv-untreatedcells, newly synthesized full-length E-cadherin appeared 1 h afterenzyme treatment and disappeared after 4 h, suggesting that newlysynthesized E-cadherins in dissociated hESCs were not stable. However,in Tzv-treated cells, E-cadherin expression was significantly increased(FIG. 2g ). Furthermore, flow cytometry analysis revealed that cellsurface E-cadherins in hESCs were significantly increased by Tzv (FIG.2h ). Therefore. Tzv is likely to affect cell adhesion by modulatingcell surface level of E-cadherins. Semiquantitative RT-PCR revealedcomparable amounts of E-cadherin transcripts in mock controls andTzv-treated cells (FIG. 2i ), suggesting the difference in E-cadherinprotein levels was not due to altered transcription levels. It is likelythat Tzv exerts its effect through stabilization of E-cadherin on thecell surface. Finally, endocytosis assay revealed that internalizationof E-cadherins was significantly blocked by Tzv. These results indicatethat Tzv regulates E-cadherin activities through inhibition ofendocytosis of E-cadherins (FIG. 2j ).

Cell-cell dissociation by trypsin leads to rapid cleavage and subsequentdestabilization of E-cadherins. We hypothesized that E-cadherinstability might also be mediated by its homophilic-interaction betweenthe cells. Thus homophilic ligation of E-cadherins with recombinantligands may stabilize E-cadherins and affect hESC survival. To test thishypothesis, we coated plates with a dimeric E-cadherin-Fc chimeraprotein containing the E-cadherin ectodomain fused to the IgG Fcfragment (Ecad-Fc). Remarkably, dissociated hESCs attached to the coatedsurface and their survival rate was significantly increased in a dosedependent manner (FIG. 2k ), confirming that cell-cell adhesion mediatedby E-cadherin is an important regulator for hESC survival.

Both Tzv and Ptn have dramatic effect on survival of hESCs grown onMatrigel-coated plates. Such survival promoting effect seems unlikelydue to influence on cell growth and may be largely attributed to theincrease in cell adhesion ability following cell dissociation andseeding processes (FIG. 3a,b ). Indeed, dissociated hESCs that weretreated with Tzv or Ptn displayed a dramatically increased adhesion toMatrigel or laminin. In contrast, hESCs' adhesion to gelatin orpoly-lysine (FIG. 3b and data not shown), which does not involveintegrins, was not affected by Ptn or Tzv treatment. The main componentof Matrigel is laminin, and it was reported that laminin receptor β1integrin is highly expressed in hESCs (Xu, C. et al., Nat Biotechnol 19(10):971-974 (2001)). To test whether Ptn or Tzv acts through β1integrin, we pretreated cells with a blocking antibody against β1integrin, and observed that the increased cell attachment induced bycompound treatment was completely abolished. This suggests that Tzv andPtn mediate cell adhesion to ECM substrates through β1 integrin (FIG. 3c).

To gain insights into the mechanism of β1 integrin regulation by Tzv andPtn, we investigated whether the compounds' effect is due to changes ofintegrin expression. In contrast to E-cadherin, β1 integrin was notcleaved by trypsin. Western blot analysis revealed that the compounds'effect was unlikely due to increased expressions of β1 integrin. Thus.Tzv and Ptn are likely to affect cell adhesion by modulating integrinactivity (FIG. 3 d,e). To examine the effects of compound treatment onthe activity of β1 integrin, we used the monoclonal antibody HUTS-21,which specifically binds to the activated form of the β1 integrin(Luque, A. et al., J Biol Chem 271 (19):11067-11075 (1996)). Notably,compounds treatment increased the level of HUTS-21 binding (FIG. 3f,g ).These results collectively suggest that Tzv and Ptn increase celladhesion by the inside-out modulation of integrin activity.

If both chemicals did enhance cell adhesion by converting integrins intoan active conformation, treatment of cells with the integrin-activatingantibody, which locks integrins in an active conformation, should have asimilar effect as compounds. Indeed, when dissociated hESCs were platedon laminin in the presence of TS2/16, an activating antibody to β1integrin (van de Wiel-van Kemenade. E. et al. J Cell Biol 117(2):461-470 (1992)), cell adhesion was significantly increased and cellsformed an increased number of colony as compared to control (FIG. 3h,i). These results suggest that the increased adhesion, which occurs whencells are treated with these compounds, involves a mechanism thatinduces integrin activation.

To further dissect the molecular mechanism by which Tzv and Ptn regulateintegrin activity, we examined the effects of several pathwayinhibitors. We found that bisindolylmaleimide I, a specific inhibitor ofPKC, could antagonize the increased cell adhesion induced by Ptn, buthad no effect on cell adhesion induced by Tzv. This suggests that PKCmay mediate the action of Ptn but not Tzv (FIG. 3j ). To further confirmthe role of PKC on hESC survival, dissociated hESCs were treated withPKC agonist phorbol 12-myristate 13-acetate (PMA). Treatment of PMAcaused integrin activation, as well as a substantial increase in celladhesion and colony formation (FIG. 3 k,l,m).

Stem cell fate is influenced by its cellular niche, which consists ofgrowth factors, cell-ECM interaction, and cell-cell interaction. Thefact that hESC survival is highly dependent on cell-cell interactionand/or cell-ECM interaction, revealed the importance of such previouslyunrecognized in vitro niche for hESCs. More importantly, cell intrinsicprotein expressions (e.g. E-cadherins and integrins) and regulatorymechanisms (e.g. protein stabilization and activation), not only respondto, but also are essential niche components, suggesting stem cellspossess intrinsic ability to construct their own niche in the absence ofother extrinsic factors or cell types, which however can participate andenhance cells' auto-regulatory niche mechanism.

Interplay between physical/structural environment and growth factorplays a very important role in cell fate regulation (Comoglio, P. M.,Boccaccio, C., & Trusolino, L., Curr Opin Cell Biol 15 (5):565-571(2003)). To examine whether growth factors are involved inintegrin-mediated hESC survival, we treated dissociated hESCs with Tzvor Ptn together with individual highly specific growth factor receptorinhibitors. We found that chemical inhibition of FGFR, IGFR, EGFR1 orErb2 greatly diminished survival promoting effect induced by Tzv or Ptntreatment (FIG. 4a ). In addition, Ptn significantly increased thephosphorylation of growth factor receptor, suggesting that engagement ofgrowth factor receptors is required for integrin-mediated cell survival(FIG. 4b ). Similarly inhibition of FGFR, IGFR, EGFR1 or Erb2 alsogreatly abolished Tzv-induced hESC survival in suspension culture.Furthermore, Tzv induced binding of E-cadherins to EGFR1 and ERB2,indicating the important role of growth factor receptors inE-cadherin-mediated cell survival (FIG. 4c, d ).

Phosphatidylinositol-3-kinase (PI-3K) signaling and MAPK/ERK are majorregulators for hESC self-renewal (Armstrong, L. et al., Hum Mol Genet 15(11):1894-1913 (2006); Paling. N. R. et al., J Biol Chem 279(46):48063-48070 (2004); Pyle, A. D., Lock, L. F., & Donovan, P. J., NatBiotechnol 24 (3):344-350 (2006); Li, J. et al., Differentiation 75(4):299-307 (2007)). Phosphorylation of ERK and AKT, a downstreameffector of PI-3K, were increased upon treatment of dissociated hESCswith Ptn, and this increase was abolished by integrin blocking antibody(FIG. 4e,f ). Moreover, activation of AKT and ERK by Ptn was blocked byinhibitors of FGFR, IGFR, EGFR or Erb2 (FIG. 4g and data not shown).Chemical inhibition of PI-3K action significantly antagonized survivaleffect induced by Ptn (FIG. 4h ). Inhibition of ERK did not have adramatic effect on survival induced by Ptn but induced hESCdifferentiation (FIG. 4i ). These results demonstrated that activationof PI-3K is a major survival signaling and activation of ERK is ananti-differentiation signaling generated by the niche through activationof growth factor receptors.

In summary, we identified two novel synthetic small molecules withdistinct mechanisms of action from a high throughput phenotypic screenthat greatly enhance hESC survival after single cell dissociation. Suchchemical tools and newly identified biological tools through mechanisticcharacterizations (e.g. defined recombinant Ecad-Fc for hESC attachmentin adherent culture; activating antibodies for enhanced cell survivaland attachment) would enable more robust hESC culture and significantlyfacilitate applications of hESCs such as gene targeting or drugdiscovery. More importantly, in-depth mechanistic characterizationsuncovered previously unrecognized niche mechanisms that are required tosustain hESC survival and proliferation. Such niche consists ofE-cadherin-mediated interaction between hESC themselves,integrin-mediated cell-ECM interaction, and growth factors. Earlierstudies have pointed to an important role of growth factors on hESCself-renewal. However, full activation of growth factor signalingrequires not only the presence of the growth factors and receptors butalso an interaction with a particular microenvironment. When thisphysical/structural environment is destroyed, growth factors alone arenot sufficient for self-renewal of ESCs.

Recently, it was reported that differentiated fibroblasts generated fromhESCs in self-renewal culture created an in vitro niche for hESCs(Bendall, S. C. et al., Nature 448 (7157):1015-1021 (2007)). Under ourand others' chemically defined medium conditions, we very rarely observesuch differentiated cells in long-term culture, suggesting that suchartificial niche might be created due to the media differences.Nevertheless, our studies reveal unique cell-autonomous (i.e. cell-cellinteraction) and non cell-autonomous (i.e. cell-ECM and -growth factor)niche mechanisms for hESC survival and self-renewal, which may likelyplay important roles in controlling adult stem cell fate in vivo.

Cell-cell dissociation by trypsin led to not only de-stabilization ofE-cadherins but also inactivation of integrins, indicating thatsignaling which maintains integrin activity is sensitive to enzymatictreatment. Feeder cell-conditioned media (with growth factor-rich serum)didn't provide much protection against cell death after single celldissociation. In addition, the fact that high density cell seeding alsoinduces an increase in cell adhesion/survival suggests that signalingrequired to maintain integrin activity may not come from secretedfactors but instead from physical cell-cell interactions. Tzv inhibitsendocytosis of E-cadherin, and thus protects cells from death insuspension. Similarly, by inhibiting endocytosis, Tzv may maintainintegrin activity by stabilizing signaling from the cell surface. On theother hand, Ptn may mimic the downstream signaling from physicalcell-cell interaction to activate PKC. Future target identification ofPtn may shed new light on the mechanism by which cell-cell adhesionregulates cell-ECM interaction. Our research also exemplified thefeasibility and advance of high throughput chemical screening in stemcell studies. Further development and application of such chemicalapproach in stem cells will undoubtedly lead to the identification ofadditional novel small molecules and mechanistic insight for preciselycontrolling cell fate in vitro and in vivo.

Methods

Cell Culture

Human ESC lines H1, HUES7 and HUES9 were cultured on irradiated MEFfeeder cells in DMEM-F12 supplemented with 2 mM L-glutamine, 1×nonessential amino acids, 20% serum replacement (Invitrogen) and 10ng/ml basic Fibroblast growth factor (Invitrogen). Chemically-definedand feeder-free hESC culture was described previously (Yao, S. et al.,Proc Natl Acad Sci USA 103 (18):6907-6912 (2006)). Briefly, hESCs weregrown on Matrigel-coated tissue culture plates in N2B27-CDM (DMEM-F12supplemented with 1×N2 supplements, 1×B27 supplements, 2 mM L-glutamine,0.11 mM 2-mercaptoethanol, 1× nonessential amino acids, and 0.5 mg/mlBSA (fraction V)) and 20 ng/ml bFGF. Human ESCs were passaged every 5-6days with 0.05% trypsin.

For clonal survival assays, single hESCs were diluted to clonal densityand plated onto 96-well Matrigel-coated plate. For low-density survivalassays, 500 cells were plated onto 96-well Matrigel-coated plate. Tovisualize hESC colonies, cultures were fixed in 4% paraformaldehyde inPBS for 5 min, washed once in PBS, then stained for alkaline phosphataseactivity as described in manufacturer's instructions. ALP positivecolonies were counted on an inverted microscope.

Reagents

ALP detection kit and integrin antibodies were from Chemicon. AG825(Erb2 inhibitor), AG1478 (EGFR inhibitor), PPP (IGFR1 inhibitor) werepurchased from Calbiochem. Antibodies raised against theintracytoplasmic tail of E-cadherins (Transduction Laboratories,Lexington, Ky.) were used for immunoprecipitation. The antibody TS2/16was from Pierce. Antibodies to the extracellular domain of E-cadherinmolecule were from Zymed (Carlsbad). Antibodies against extracellularsignal-regulated kinase/MAPK, EGFR1, ERB2, GADPH and phosphorylated formof AKT were from Cell Signaling. Mouse monoclonal anti-phosphotyrosine(4G-10 clone) was from Upstate Biotechnology. Ptn and Tzv were added toculture medium at 2 μM.

High-Throughput Chemical Screen.

The trypsinable hESC lines HUES7 or HUES9 were used for the screen.hESCs were cultured in chemically-defined media on the Matrigel-coatedplate as described above. Then cells were harvested by trypsin. hESCswere plated at 4,000 cells per well onto Matrigel-coated 384-wellplates. After 1 h when cells settled down, compounds from a library of50,000 discrete heterocycles were added to each well (2 μM finalconcentration). After an additional 6 days of incubation, in which mediaand compounds were changed at day 3, cells were stained for ALPexpression and examined for compact colony morphology.

Immunostaining Analysis.

Immunostaining was performed as described previously (Yao, S. et al.,Proc Natl Acad Sci USA 103 (18):6907-6912 (2006)). Briefly, cells werefixed with 4% paraformaldehyde at mom temperature (RT) for 15 min. Thecells were then incubated at RT in blocking buffer for 1 hour. Primaryantibody incubation was carried overnight at 4° C. The followingcommercially available antibodies were used at a concentration of 1:100in blocking buffer: anti-SSEA4, anti-Oct4 (Chemicon??) anti-Nanog(Chemicon). The staining was visualized using secondary antibodiesconjugated to FITC, cy3 or cy5 (Jackson ImmunoResearch).

Teratoma Formation and Karyotyping.

Teratoma formation experiments were performed by injecting 3-5 millionhESCs (maintained in the presence of compounds Tvz or Ptn) under thekidney capsule of nude mice. After 4-5 weeks, all mice developedteratomas, which were removed and then immunohistologically analyzed byThe Scripps Research Institute Research Histology Service and AnimalResources. Compounds treated cells were karyotyped by standard G-bandingat the Children's Hospital Oakland. Cytogenetics Laboratory. Nochromosomal abnormality was found in the 10 randomly picked nuclei.

TUNEL Assay

The hESCs under different treatments were dissociated by trypsin andfixed by 4% paraformaldehyde. And the staining was carried out accordingto the manufacturer's instructions (MBL Laboratories, Watertown, Mass.).After staining, samples were analysed by flow cytometry using a FACSCalibur flow cytometer (BD).

Flow Cytometry Analysis

To assess the expression of E-cadherin, activated integrin and SSEA4,dissociated cells (3×10⁵) were washed with PBS and resuspended in PBScontaining 2% goat serum. Cells were then incubated with the appropriateantibody for 1 h at 4° C., washed with the blocking solution, andlabeled with FITC-conjugated secondary antibody for 30 min at 4° C.Cells were then washed and analyzed on a FACS Calibur flow cytometer.

Cell Adhesion Assay

Cell adhesion assays were performed in 96-well microtiter plates coatedwith Matrigel. After trypsin, hESCs were resuspended in thechemically-defined media containing the desired compounds. Cells werethen added to the microtiter wells and incubated for 3 h at 37° C.Unbound and loosely bound cells were removed by shaking and washing, andthe remaining cells were then fixed immediately. The wells were washed 3times with 200 μl of H₂O, and attached cells were stained with CrystalViolet (Sigma). The absorbance of each well at 570 nm was then measured.For experiments with blocking antibodies, cells were pre-incubated withantibodies on ice for 30 min, and adhesion assays were performed in thepresence of antibodies. Each sample was assayed independently for threetimes.

Endocytosis Assay

hESCs were incubated with 1.5 mg/ml sulfosuccinimidyl 2-(biotinamido)ethyl-dithioproprionate (sulfo-NHS-SS-biotin) (Pierce Chemical Co.) onice, followed by washing and quenching. Endocytosis of E-cadherin wasinitiated by Ca²⁺ depletion and 37° C. incubation. Cells were thenincubated in two 20-min washes of glutathione solution (60 mMglutathione, 0.83 M NaCl, with 0.83 M NaOH and 1% BSA added before use)at 0° C. which removed all cell surface biotin groups. Remainingbiotinylated proteins were sequestered inside cells by endocytosis andwere therefore protected from glutathione stripping. Biotinylatedproteins were recovered on streptavidin beads and analyzed by SDS-PAGE.E-Cadherins were detected by immunoblotting. Total level of surfaceE-cadherin before endocytosis was used as reference.

Example 2 Synthesis ofN-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide (Thiazovivin)

Chemical Synthesis

Using the chemical synthesis examples presented below and chemicalsynthesis methods generally known in the art, one of skill is capable ofmaking the compounds disclosed herein (e.g. the compounds of Formulae(I) to (VI)).

All chemicals obtained commercially were used without furtherpurification. NMR spectra were recorded on a Bruker (400 MHz)instrument. Chemical shifts (b) were measured in ppm and couplingconstants (J) are reported in Hz. LCMS was performed by reverse-phaseliquid chromatography-mass spectrometer Agilent 1100 LCMS system withAPI-ES ionization source. High pressure liquid chromatography wasperformed with C18 column with a linear gradient from 10% solvent A(acetonitrile with 0.035% trifluoroacetic acid) in solvent B (water with0.05% trifluoroacetic acid) to 90% A in seven and half minutes, followedby two and half minutes elution with 90% A.

Synthesis of N-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide(Thiazovivin)

Benzyl amine was loaded to 4-formyl-3,5-dimethoxyphenoxymethylfunctionalized polystyrene resin (PAL) via reductive amination to givePAL-benzyl amine resin. See, Ding. S.; Grey, N. S. Wu, X.; Ding, Q.;Schultz, P. G. J. Am. Chem. Soc. 2002, 124, 1594-1596. A reaction flaskcontaining PAL-benzyl amine resin (200 mg, 0.2 mmol),2-bromothiazole-4-carboxylic acid (83 mg, 0.4 mmol),bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) (153 mg, 0.6 mmol)and diisopropylethylamine (0.17 mL, 1 mmol) in DMF (3 mL) was shaken for24 hr at room temperature. The resin was washed with methanol,dichloromethane and dried in vacuo to give PALresin-N-benzyl-2-bromothiazole-4-carboxamide, which was then added to aflame-dried reaction vial, followed by 4-aminopyrimidine (95 mg, 1mmol), Pd₂(dba)₃ (46 mg, 0.05 mmol), Xantphos (87 mg, 0.15 mmol) andNaO^(t)Bu (192 mg, 2 mmol). The vial was sure safe capped and degassed,then charged with argon and anhydrous dioxane (1.5 mL). The reaction wasshaken for 24 hours at 90° C. The resin was washed with sodiumdiethyldithiocarbamate solution (0.05 M in DMF), methanol anddichloromethane and dried in vacuo. The resin was subsequently cleavedwith cleavage cocktail TFA:CH₂Cl₂:H₂O (45:55:5) (2 mL) for 2 hr. Theresin was filtered, the filtrate was collected and evaporated in vacuoto give the crude which was then purified by HPLC to give the titlecompound (30 mg, 48%).

N-Benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide

Exact mass calculated for C₁₅H₁₃N₅OS: 311.1. found LCMS m/z=334.1(M+Na⁺).

¹H NMR (400 MHz, d₆-DMSO) 4.49 (d, J=6.3 Hz, 2H), 5.76 (s, 1H),7.21-7.27 (m, 2H), 7.30-7.34 (m, 4H), 7.85 (s, 1H), 8.45 (t, J=6.3 Hz,1H), 8.51 (d, J=6.1 Hz, 1H), 8.94 (s, 1H).

Example 3 Synthesis of Thiazovivin Derivatives

Appropriate amines R¹NH₂ were pre-loaded to4-formyl-3,5-dimethoxyphenoxymethyl functionalized polystyrene resin(PAL) via reductive amination to give PAL-benzyl amine resin. A mixtureof PAL-benzyl amine resin (200 mg, 0.2 mmol, 1.0 eq.), 2-bromothiazolecarboxylic acid 1 (0.4 mmol, 2.0 eq.),bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) (0.6 mmol, 3.0eq.) and diisopropylethylamine (1 mmol, 5.0 eq.) in anhydrous DMF (3 mL)was shaken for 24 hr at ambient temperature. The resin was washed withmethanol, dichloromethane and dried in vacuo, which was then added to aflame-dried reaction vial, followed by corresponding R²NH₂ (1 mmol, 5.0eq.), Pd₂(dba)₃ (0.05 mmol), Xantphos (0.15 mmol) and NaO^(t)Bu (2 mmol,10.0 eq.). The vial was sure safe capped and degassed, then charged withargon and anhydrous dioxane (1.5 mL). The reaction was shaken for 24hours at 90° C. The resin was washed with sodium diethyldithiocarbamatesolution (0.05 M in DMF), methanol and dichloromethane and dried invacuo. The resin was subsequently cleaved with cleavage cocktail:TFA:CH₂Cl₂:H₂O=45:55:5 (2 mL) for 2 hr. The resin was filtered and thefiltrate was collected and evaporated in vacuo to give the crude whichwas then purified by HPLC to give the desired title compound 3.

Structure Name Data

N-benzyl-2-(pyrimidin-4- ylamino)thiazole-4-carboxamide LC/MS Rt = 1.49min, [MH⁺] 312, [MNa⁺] 334.

N-benzyl-2-(6- methoxypyrimidin-4- ylamino)thiazole-4-carboxamide LC/MSRt = 2.12 min, [MH⁺] 342.

N-benzyl-2- (phenylamino)thiazole-4- carboxamide LC/MS Rt = 2.45 min,[MH⁺] 310.

N-benzyl-2-(pyridin-2- ylamino)thiazole-4-carboxamide LC/MS Rt = 1.69min, [MH⁺] 311.

N-benzyl-2-(pyridin-4- ylamino)thiazole-4-carboxamide LC/MS Rt = 1.57min, [MH⁺] 311.

N-benzyl-2-(pyrazin-2- ylamino)thiazole-4-carboxamide LC/MS Rt = 2.02min, [MH⁺] 312.

N-benzyl-5-isopropyl-2- (pyrimidin-4-ylamino)thiazole- 4-carboxamideLC/MS Rt = 1.90 min, [MH⁺] 354.

N-(pyridin-3-ylmethyl)-2- (pyrimidin-4-ylamino)thiazole- 4-carboxamideLC/MS [MH⁺] 312.

2-(pyrimidin-4-ylamino)-N-(3- (trifluoromethyl)benzyl)thiazole-4-carboxamide LC/MS [M⁺] 379.

N-(4-methoxyphenethyl)-2- (pyrimidin-4-ylamino)thiazole- 4-carboxamideLC/MS [M⁺] 355.

N-(benzo[d][1,3]dioxol-5- ylmethyl)-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide LC/MS [MH⁺] 356.

methyl 4-((2-(pyrimidin-4- ylamino)thiazole-4-carboxamido)methyl)benzoate LC/MS [MH⁺] 370.

4-((2-(pyrimidin-4- ylamino)thiazole-4- carboxamido)methyl)benzoic acidLC/MS [MH⁺] 356.

N-(4-(butylcarbamoyl)benzyl)- 2-(pyrimidin-4-ylamino)thiazole-4-carboxamide LC/MS [MH⁺] 411.

N-(4- dimethylcarbamoyl)benzyl)-2- (pyrimidin-4-ylamino)thiazole-4-carboxamide LC/MS [MH⁺] 383.

N-(4-methoxybenzyl)-2- (pyrimidin-4-ylamino)thiazole- 4-carboxamideLC/MS [MH⁺] 342.

N-(3-methoxybenzyl)-2- (pyrimidin-4-ylamino)thiazole- 4-carboxamideLC/MS [MH⁺] 342.

N-(4-morpholinophenyl)-2- (quinolin-8-ylamino)thiazole-5- carboxamideLC/MS [MH⁺] 432.

Example 4 Synthesis ofN-(cyclopropylmethyl)-4-(4-(6-hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidin-2-ylamino)benzenesulfonamide(Pyrintegrin)

The reaction flask containing 2,4-dichloropyrimidine (372 mg, 2.5 mmol),6-methoxy-1,2,3,4-tetrahydroquinoline (489 mg, 3 mmol) anddiisopropylethylamine (0.52 mL, 3 mmol) in n-butanol (10 mL) was heatedat 40° C. overnight. The solvent was evaporated, and the residue waspurified by flash column chromatography to give2-Chloro-4-(6-methoxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidine (551 mg,80%). This intermediate (250 mg, 0.91 mmol) was then dissolved indichloromethane and treated with BBr₃ (1 M in dichloromethane) (1 mL, 1mmol) at −78° C. The reaction mixture was slowly warmed up to roomtemperature and stirred for 1 hr, poured into water, extracted withdichloromethane. The combined organics were dried over anhydrous Na₂SO₄and concentrated. The residue was purified by flash columnchromatography to give2-Chloro-4-(6-hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidine (154 mg,65%). To a stirred solution of2-chloro-4-(6-hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidine (29 mg,0.11 mmol) and 4-amino-N-(cyclopropylmethyl)benzenesulfonamide (27 mg,0.12 mmol) in DMF (0.5 mL) was added p-toluenesulfonic acid (2 M indioxane) (55 μL, 0.11 mmol). The reaction mixture was stirred at 90° C.overnight, then purified by HPLC to give the title compound (27 mg,56%).

N-(Cyclopropylmethyl)-4-(4-(6-hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidin-2-ylamino)benzenesulfonamide

Exact mass calculated for C₂₃H₂₅N₅O₃S: 451.2. found LCMS m/z=452.3(M+H⁺).

¹H NMR (400 MHz, d₆-DMSO) 0.05-0.09 (m, 2H), 0.32-0.36 (m, 2H),0.75-0.81 (m, 1H), 1.90-1.95 (m, 2H), 2.64 (t, J=6.4 Hz, 4H), 3.93 (t,J=6.5 Hz, 2H), 6.59 (d, J=7.1 Hz, 1H), 6.66-6.70 (m, 2H), 7.25-7.28 (m,1H), 7.64 (t, J=5.9 Hz, 1H), 7.74 (d, J=8.8 Hz, 2H), 7.82 (d, J=8.8 Hz,2H), 8.01 (d, J=7.1 Hz, 1H), 10.79 (s, 1H).

Example 5 Synthesis of Pyrintegrin Derivatives

To a mixture of 2,4-dichloropyrimidine 4 (1.0 eq.), R¹NH₂ (1.2 eq.) anddiisopropylethylamine (1.2 eq.) in n-butanol was heated at 80° C.overnight. The solvent was evaporated, and the residue was purified byflash column chromatography to intermediate 10 in excellent yield(>80%), which was then treated with R²NH₂ (1.2 eq.) in DMF was addedp-toluenesulfonic acid (2 M in dioxane) (1.2 eq.). The reaction mixturewas stirred at 90° C. overnight, and then purified directly bypreparative HPLC to give Pyrintegrin derivatives 11 in excellent yields.

Structure Name Data

N-(cyclopropylmethyl)-4-(4-(6- hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidin-2- ylamino)benzenesulfonamide LC/MS [MH⁺] 452.

N-(cyclopropylmethyl)-4-(4-(6- hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidin-2- ylamino)benzamide LC/MS [MH⁺] 416.

N-cyclopropyl-4-(4-(6-hydroxy- 3,4-dihydroquinolin-1(2H)-yl)pyrimidin-2- ylamino)benzenesulfonamide LC/MS [MH⁺] 438.

N-cyclopropyl-4-(4-(6-hydroxy- 3,4-dihydroquinolin-1(2H)-yl)pyrimidin-2- ylamino)benzamide LC/MS [MH⁺] 402.

4-(4-(6-hydroxy-3,4- dihydroquinolin-1(2H)- yl)pyrimidin-2-ylamino)-N-isobutylbenzenesulfonamide LC/MS [MH⁺] 468.

4-(4-(6-hydroxy-3,4- dihydroquinolin-1(2H)- yl)pyrimidin-2-ylamino)-N-methylbenzenesulfonamide LC/MS [MH⁺] 412.

4-(4-(6-hydroxy-3,4- dihydroquinolin-1(2H)- yl)pyrimidin-2-ylamino)-N-isopentylbenzamide LC/MS [MH⁺] 432.

4-(4-(6-hydroxy-3,4- dihydroquinolin-1(2H)- yl)pyrimidin-2-ylamino)-N-methylbenzamide LC/MS [MH⁺] 376.

4-(4-(6-hydroxy-3,4- dihydroquinolin-1(2H)- yl)pyrimidin-2-ylamino)benzenesulfonamide LC/MS [MH⁺] 398.

1-(2-(4- phenoxyphenylamino)pyrimidin-4-yl)-1,2,3,4-tetrahydroquinolin-6- ol LC/MS [MH⁺] 411.

1-(2-phenylamino)pyrimidin-4- yl)-1,2,3,4-tetrahydroquinolin-6-ol LC/MS[MH⁺] 319.

4-(4-(6-hydroxy-3,4- dihydroquinolin-1(2H)-yl)pyrimidin-2-ylamino)-N-(2- hydroxyethyl)benzenesulfonamide LC/MS[MH⁺] 442.

N-isopentyl-4-(4-(6-methoxy-3,4- dihydroquinolin-1(2H)- yl)pyrimidin-2-ylamino)benzenesulfonamide LC/MS [MH⁺] 482.

N-isopentyl-4-(4-(6-methoxy-3,4- dihydroquinolin-1(2H)- yl)pyrimidin-2-ylamino)benzamide LC/MS [MH⁺] 446.

4-(4-(6-methoxy-3,4- dihydroquinolin-1(2H)- yl)pyrimidin-2-ylamino)-N-methylbenzamide LC/MS [MH⁺] 390.

4-(4-(6-methoxy-3,4- dihydroquinolin-1(2H)- yl)pyrimidin-2-ylamino)-N-methylbenzenesulfonamide LC/MS [MH⁺] 423.

isopropyl 2-(2-(4- sulfamoylphenylamino)pyrimidin- 4-ylamino)benzoateLC/MS [MH⁺] 428.

isopropyl 2-(2-(4-N- isopentylsulfamoyl)phenylamino)-pyrimidin-4-ylamino)benzoate LC/MS [MH⁺] 498.

N-isopentyl-4-(4- morpholinopyrimidin-2- ylamino)benzenesulfonamideLC/MS [MH⁺] 406.

4-(3,4-dihydroisoquinolin-2(1H)- yl)-N-phenylpyrimidin-2-amine LC/MS Rt= 2.03 min, [MH⁺] 303.

4-(3,4-dihydroisoquinolin-2(1H)- yl)-N-(4- morpholinophenyl)pyrimidin-2-amine LC/MS Rt = 1.84 min, [MH⁺] 388.

4-(3,4-dihydroisoquinolin-2(1H)- yl)-N-(3,4,5-trimethoxyphenyl)pyrimidin-2- amine LC/MS Rt = 1.95 min, [MH⁺] 393.

N1-(4-(3,4-dihydroisoquinolin- 2(1H)-yl)pyrimidin-2-yl)-N4,N4-dimethylbenzene-1,4-diamine LC/MS Rt = 1.49 min, [MH⁺] 346.

4-(3,4-dihydroisoquinolin-2(1H)- yl)-N-(3-phenoxyphenyl)pyrimidin-2-amine LC/MS Rt = 2.36 min, [MH⁺] 395.

4-(3,4-dihydroisoquinolin- 2(1H)-yl)pyrimidin-2- yl)isoquinolin-6-amineLC/MS Rt = 1.51 min, [MH⁺] 354.

N-(4-(3,4-dihydroisoquinolin- 2(1H)-yl)pyrimidin-2-yl)-1H- indol-5-amineLC/MS Rt = 1.96 min, [MH⁺] 342.

N-(3-(4-(3,4-dihydroisoquinolin- 2(1H)-yl)pyrimidin-2-ylamino)phenyl)acetamide LC/MS Rt = 1.80 min, [MH⁺] 360.

4-(3,4-dihydroisoquinolin-2(1H)- yl)-N-(pyridin-2-yl)pyrimidin-2- amineLC/MS Rt = 1.95 min, [MH⁺] 304.

4-(3,4-dihydroisoquinolin-2(1H)- yl)-N-(pyridin-3-yl)pyrimidin-2- amineLC/MS Rt = 1.42 min, [MH⁺] 304.

4-(3,4-dihydroisoquinolin-2(1H)- yl)-N-(pyridin-4-yl)pyrimidin-2- amineLC/MS Rt = 1.40 min, [MH⁺] 304.

N-(4-(3,4-dihydroisoquinolin- 2(1H)-yl)pyrimidin-2-yl)isoquinolin-1-amine LC/MS Rt = 2.20 min, [MH⁺] 354.

4-(3,4-dihydroisoquinolin-2(1H)- yl)-N-(pyrimidin-2-yl)pyrimidin-2-amine LC/MS Rt = 1.70 min, [MH⁺] 305.

4-(3,4-dihydroisoquinolin-2(1H)- yl)-N-(pyrazin-2-yl)pyrimidin-2- amineLC/MS Rt = 1.56 min, [MH⁺] 305.

4-(3,4-dihydroisoquinolin-2(1H)- yl)-N-(1-methyl-1H-pyrazol-5-yl)pyrimidin-2-amine LC/MS Rt = 1.64 min, [MH⁺] 307.

4-(4-morpholinopiperidin-1-yl)-N- phenylpyrimidin-2-amine LC/MS Rt =0.97 min, [MH⁺] 340.

4-(4-morpholinopiperidin-1-yl)-N- (3,4,5- trimethoxyphenyl)pyrimidin-2-amine LC/MS Rt = 1.11 min, [MH⁺] 430.

N1,N1-dimethyl-N4-(4-(4- morpholinopiperidin-1-yl)pyrimidin-2-yl)benzene-1,4- diamine LC/MS [MH⁺] 383

4-(4-morpholinopiperidin-1-yl)-N- (3-phenoxyphenyl)pyrimidin-2- amineLC/MS [MH⁺] 379

N-(4-4-morpholinopiperidin-1- yl)pyrimidin-2-yl)isoquinolin-6- amineLC/MS [MH⁺] 391

N-(4-4-morpholinopiperidin-1- yl)pyrimidin-2-yl)-1H-indol-5- amine LC/MS[MH⁺] 379

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A compound having the formula:

wherein, R²⁰ is substituted C₁₋₆ alkyl, or unsubstituted C₃₋₈cycloalkyl, wherein the substituted C₁₋₆ alkyl is substituted with C₃₋₈cycloalkyl; R²⁸ is —OR⁴⁰; and R⁴⁰ is hydrogen, or unsubstituted C₁₋₆alkyl; or a racemate, diastereomer, tautomer, or a geometric isomerthereof, or a pharmaceutically acceptable salt thereof.
 2. The compoundof claim 1 having the formula:

or a racemate, diastereomer, tautomer, or a geometric isomer thereof, ora pharmaceutically acceptable salt thereof.
 3. The compound of claim 1having the formula:

or a racemate, diastereomer, tautomer, or a geometric isomer thereof, ora pharmaceutically acceptable salt thereof.
 4. A composition comprisinga population of isolated cells comprising an amount of a compound ofclaim 1 sufficient to improve survival of isolated cells by at least2-fold compared to the absence of the compound.
 5. The composition ofclaim 4, wherein the cells are selected from the group consisting ofstem cells, induced stem cells, pluripotent stem cells, progenitorcells, differentiated cells, beta cells and fibroblasts.